Endophytic bacteria from in vitro culture of Leucojum aestivum L. a new source of galanthamine and elicitor of alkaloid biosynthesis

Leucojum aestivum is known for its ability to biosynthesize alkaloids with therapeutic properties, among which galanthamine used for the treatment of Alzheimer's disease. New sources of this alkaloid are still being explored. In this study, a novel strain PLV of endophytic bacterium Paenibacillus lautus was isolated from in vitro L. aestivum plants. We report the whole genome sequence of that strain and its capacity to produce alkaloids and growth regulators. The effect of elicitation with autoclaved bacteria on the production of alkaloids was examined. Ten alkaloids were identified in bacteria extracts: galanthamine, lycorine, ismine, lycoramine, haemanthamine, tazettine, galanthine, homolycorine, 1,2-dihydrochlidanthine, and hippeastrine. The mean contents of galanthamine and lycorine were 37.51 µg/g of dry weight (DW) and 129.93 µg/g of DW, respectively. Moreover, isolated P. lautus strain synthesized: indole-3-acetic acid, t-zeatin, c-zeatin, kinetin, gibberellin A1, abscisic acid, salicylic acid, benzoic acid. In vitro elicitation of cultures with P. lautus increased dry biomass, stimulated galanthamine and lycorine production, contributed to 8,9-desmethylenebis (oxy)-7,9 dimethoxy-crinan biosynthesis, change pigments content, and antioxidant enzymes activities. Our findings for the first time point out that galanthamine can be synthesized by an microorganism. Moreover isolated strain can be used as a new elictor of Amaryllidaceae alkaloids biosynthesis.


Results and discussion
Isolation and molecular identification of endophytic bacteria from in vitro L. aestivum plants. Endophytic bacteria are often observed in in vitro plant cultures. They are known to live inside plant tissues without the plants exhibiting symptoms of disease 27 . In this study, no significant outgrowth of bacteria was observed during micropropagation of L. aestivum plants (see Supplementary Fig. S1a). The only indication of the presence of bacteria was a slight smear around the bulbs or roots. However, increased bacterial growth was noted after transferring the L. aestivum culture from 5 to 25 °C to the medium containing melatonin. The endophytic bacterium described in this study was isolated from leaf and bulbs fragments of in vitro-grown L. aestivum and cultured on solidified lysogeny broth medium (LB) 28 (see Supplementary Fig. S1b, c). Bacterial colonies were classified as Gram positive and further identified by whole genome sequence analysis. Genome sequencing of the isolated strain resulted in 12,903,512 paired reads of which 12,102,198 (93%) passed initial filtering. Those reads generated in total 3,6 Gb of sequence which covered Paenibacillus lautus genome 496 times on average. The total estimated genome size of isolated strain was 7,809,730 bp. Assembly and scaffolding of the reads resulted in 61 scaffolds coming form 109 cotigs with N50 of 376,288 and L50 of 7. Quality assessment for Paenibacillus lautus PLV strain assembly is presented in Supplementary Table S1. The ungapped total plasmid length was estimated at 199,621 bp.
Annotation of the assembled sequence allowed identification of 7,154 genes, of which 6,917 were protein coding genes. The genes also comprised 94 RNA genes, which included: 18 rRNA genes, (5S, 16S, 23S); 72 tRNAs and four ncRNAs. Assemblies were finally deposited in the GenBank under the Bio Project number PRJNA751742 and the strain was labeled as PLV.
Based on TYGS analysis we found that (when considering 16S rDNA sequence) the most related species/ strains were: Paenibacillus lautus (NBRC15380), Paenibacillus glucanolyticus (DSM5162), Paenibacillus lactis (DSM15596) and Paenibacillus ihbetae (JCM 31131T). Similar results were obtained when whole-genome tree was analyzed, however, Paenibacillus ihbetae was replaced by Paenibacillus solani (FJAT-22460) in the cluster of the most similar species (Fig. 1). To our knowledge, this is the first study to report the isolation of P. lautus from in vitro L. aestivum plants. Previously, Spina et al. 14 isolated bacteria belonging to the genus Bacillus from in vivo and in vitro L. aestivum bulbs.

Ability of P. lautus PLV strain to biosynthesize Amaryllidaceae alkaloids and plant growth regulators.
We demonstrated that endophytic P. lautus PLV strain isolated from in vitro L. aestivum plants synthesizes Amaryllidaceae alkaloids. To investigate the chemical composition of purified extracts, a gas chromatography-mass spectrometry (GC-MS) was used and ten alkaloids were identified (Fig. 2) by comparing the measured data with previously published data 14 and using the NIST library 31 . A detailed list of the identified alkaloids is presented in Supplementary Table S2. It is noteworthy that two of the most important alkaloids synthesized by P. lautus PLV strain were identified by comparison with authentic compounds and quantified by liquid chromatography-mass spectrometry (LC-MS): galanthamine (37.51 µg/g of DW) and lycorine (129.93 µg/g of DW) ( Table 1). The accumulation of galanthamine and lycorine by isolated bacteria is comparable to or higher than that in L. aestivum plant, shoot, and callus cultures 1,32 . Earlier reports showed the possibility of biosynthesis: lycorine, tazettine, pseudolycorine, acetylpseudolycorine, and 1,2-dihydro-chlidanthine by the endophytic bacteria of L. aestivum and lycorine, crinamidine, and powelline by the bacteria from C. macowanii 14,18 . It is worth emphasizing, that this is the first information about the synthesis of galanthamine by endophytic microorgan- Figure 1. (a) SSU tree. Tree inferred with FastME 2.1.6.1 29 from GBDP distances calculated from 16S rDNA gene sequences. The branch lengths are scaled in terms of GBDP distance formula d5. The numbers above branches are GBDP pseudo-bootstrap support values > 60% from 100 replications, with an average branch support of 76.9%. The tree was rooted at the midpoint 30 . (b) Genome tree-Tree inferred with FastME 2.1.6.1 29 from GBDP distances calculated from genome sequences. The branch lengths are scaled in terms of GBDP distance formula d5. The numbers above branches are GBDP pseudo-bootstrap support values > 60% from 100 replications, with an average branch support of 75%. The tree was rooted at the midpoint 29 . Figure 2. GC-MS total ion current chromatograms of the alkaloid fraction from Paenibacillus lautus strain PLV extract (upper trace) and alkaloids standard mixture (lower, red-shaded trace), acquired in scan mode. Identification based on library search, details given in Table S2. www.nature.com/scientificreports/ isms. It is noteworthy that P. lautus PLV strain produced 10 Amaryllidaceae alkaloids. By comparison, in vitro L. aestivum plants most often synthesize two to seven alkaloids 32,33 . Moreover, alkaloids such as galanthine and hippeastrine identified in P. lautus PLV strain extracts have not previously been isolated from L. aestivum cultures. Galanthine is an alkaloid characteristic of Galanthus woronowii 34 , among others, while hippeastrine is typical of Hippeastrum sp. 35 . It is worth noting that hippeastrine has a strong antiproliferative effect and may find potential applications in the treatment of cancer 36 . Chromatographic analyses showed that P. lautus strain PLV isolated from in vitro cultures of L. aestivum is capable of biosynthesizing auxin (indole-3-acetic acid: IAA), cytokinins (t-zeatin, c-zeatin: Z, and kinetin: K), gibberellin A 1 (GA 1 ), abscisic acid (ABA), jasmonic acid (JA), salicylic acid (SA), and benzoic acid (BeA) (Fig. 3, Supplementary Table S3). It is worth noting that the endophytic bacteria synthesized the greatest amount of stress-related hormones, such as BeA, JA and SA (6964.1, 1991.7, 771.7 ng/g DW, respectively). A slightly lower but still high amount was also reported for ABA (181.4 ng/g DW) ( Table 1). It is known that endophytic bacteria can protect their host plants from phytopathogens induced systemic resistance (ISR), therefore endophytes very often synthesize these hormones. Endophytic bacteria can initiate ISR using among others SA and JA 37 . Endogenous and exogenous jasmonates and SA regulated also the expression of the genes related to biosynthesis of secondary metabolites 38 . Perhaps the biosynthesis of Amaryllidaceae alkaloids by endophytic P. lautus

Effect of P. lautus strain PLV elicitation on dry weight increment in L. aestivum in vitro plants.
Elicitors can induce stress responses in in vitro cultures and cause morphological, physiological, biochemical, and molecular changes, thereby influencing growth. Thus, the biomasses of plant cultures may decrease after treatment with elicitors 23 . This phenomenon was not observed in our research. The dry weight (DW) of L. aestivum control and elicited with autoclaved bacteria plants increased throughout the culture period ( Table 2). The highest increase in dry biomass (0.53 g) was achieved on day 28 with the 0.04% elicitor. This increase was about 1.3 times higher than that observed in the control plants on day 28. However, on day 7, the greatest increases in dry biomass were observed in the control plants and in the plants treated with the 0.01% elicitor. On day 14, the 0.02% elicitor stimulated dry biomass accumulation in L. aestivum plants. These increases were approximately 1.89 times lower than those observed in plants treated with the 0.04% elicitor for 28 days. Thus, the stimulating effect of the elicitor could be related to the ability of P. lautus strain PLV to produce growth regulators. Endogenous growth regulators, along with exogenous ones, play a key role in in vitro morphogenesis. The most important are auxins and cytokinins. In this study, P. lautus strain PLV biosynthesized auxin (IAA) and cytokinins (t-zeatin, c-zeatin, and kinetin) ( Table 1). There is little information regarding the elicitation of tissue cultures with autoclaved endophytic bacteria. Song et al. 24 observed a stimulating effect of live endophytic bacteria on the growth of adventitious P. ginseng roots.
Effect of P. lautus strain PLV elicitation on pigments content and antioxidant enzymes activity. The analysis has proven the influence of bacterial elicitor on the production of pigments in extracts from L. aestivum plants. Elicitation of L. aestivum cultures with the 0.01% elicitor for seven days increased the chlorophyll a content about 1.6 times in comparison with control and other treated plants ( Table 2). The highest contents of chlorophyll b (25.77 µg/g of fresh weight: FW) and carotenoids (15.55 µg/g of FW) were also recorded with this concentration, but on day 28 (Table 2). Previous studies have investigated the effects of endophyte inoculation on the amounts of chlorophyll a in plants. For example, inoculation of Papaver somniferum cv. Sampada plants with a Bacillus sp. increased the chlorophyll content 44 . Root-associated endophytic fungi enhanced the chlorophyll content, photosynthesis rate, stomatal conductance, and transpiration rate of Cucumis sativus plants 45 . Inoculation of W. somnifera plants with live fungal endophytes improved their photosynthetic efficiency. The treated plants had higher chlorophyll and carotenoid amounts than control plants 25 .
Studies have shown that when plants are exposed to stressful conditions, their antioxidant systems play key protective roles by increasing the activity of antioxidant enzymes 29 . It is also known that antioxidant enzyme activity can results enhancement biosynthesis of secondary metabolites. Treatment with elicitors induces www.nature.com/scientificreports/ oxidative stress. In this study, the activities of the antioxidant enzymes catalase (CAT), peroxidase (POD), and superoxide dismutase (SOD) were examined (Fig. 4). The highest CAT activity were recorded in control plants on day 7 and in plants treated with the 0.04% elicitor on days 14 and 28 (Fig. 4a). The highest POD activity was noted on day 7 in control plants and in plants treated with the 0.02% elicitor. Slightly lower activity, but higher than under other conditions, was observed with the 0.01% and 0.04% elicitors on day 7 (Fig. 4b). This high POD activity on day 7 may have been caused by stress induced when the plants were transferred from the solid medium to the RITA ® bioreactor and were thus forced to gradually adapt to new conditions. The highest SOD activity was recorded on day 14 with the 0.01% elicitor. It is worth noting that, regardless of the conditions, SOD reached higher values on day 14 (average 1.515 U/µg of protein) than on days 7 (average 1.06 U/µg of protein) and 28 (average 0.8 U/µg of protein) (Fig. 4c). These results indicate that in L. aestivum cultures, the activity of antioxidant enzymes depends not only on the concentration of the bacterial elicitor but also on the duration of its action. In the case of L. aestivum cultures, the influence of abiotic elicitors, such as melatonin and sugars, on the activity of antioxidant enzymes has been studied. However these analyses were performed only on the 28th day of culture 10,32 .  Fig. S2, Table 3). Alkaloid biosynthesis varied according to the in vitro conditions and the culture duration (Table 3). On day 7, the 0.01% and 0.02% elicitors stimulated the biosynthesis of two alkaloids: crinan-3-ol and lycorine. By day 28, the biosynthesis of demethylmaritidine was observed both in control and in the presence of 0.01% and 0.02% elicitors while 0.04% elicitor stimulated the biosynthesis of 8,9-desmethylenebis (oxy)-7,9 dimethoxy-crinan which was not observed in any plant materials. This was consistent with our earlier observation that the use of elicitors in a culture medium stimulated the biosynthesis of alkaloids that are usually not present in in vitro L. aestivum cultures 32 . The highest number of alkaloids was observed in control plants on day 28 (galanthamine, crinan-3-ol, demethylmaritidine and lycorine). It is worth emphasizing that on the 28th day of the culture the highest activity of CAT and SOD was observed (Fig. 4). Autoclaved endophytic P. lautus strain PLV were found to increase the production of galanthamine and lycorine in L. aestivum tissue cultures (Fig. 5). It is worth noting that LC-MS used for the quantification of alkaloids from plants allowed the detection of galanthamine and lycorine in all the samples studied. The highest galanthamine content (44.47 µg/g of DW) was observed in plants treated with the 0.02% elicitor for 14 days. This content was 3.9 times higher than that in control plants that grew for 14 days (Fig. 5a). The 0.02% elicitor also stimulated lycorine biosynthesis. On day 14, a content of 235.73 µg/g of DW was recorded, compared to 145.75 µg/g of DW in control plants (Fig. 5b). These findings confirm the results of a previous study reporting that the optimum times and concentrations of abiotic elicitor treatments are important for the production of Amaryllidaceae alkaloids 39 . However, there is no information in the literature regarding the effects of autoclaved endophytic bacterial elicitation on Amaryllidaceae alkaloid biosynthesis. It should also be noted that on day 14, the 0.02% elicitor also increased the DW biomass of L. aestivum plants ( Table 2). The large increase in biomass combined with the high production of alkaloids are very important for industrial production. Liu et al. 15 showed that inoculation of in planta L. radiata with live endophytic bacteria increased the plant DW, resulting in an increase in the total yield of alkaloids. There is also little information on the use of endophytic bacteria in the biosynthesis of specialized metabolites in in vitro cultures, as endophytic fungi have typically been used for this purpose 16 . Inacio et al. 26 found that eliciting P. campestris root cultures with autoclaved endophytic B. megaterium stimulated the biosynthesis of 22 β-hydroxymaitenin. The use of live bacteria to stimulate the biosynthesis of specialized metabolites in in vitro cultures has also been reported. For example, a co-culture with endophytic bacteria increased ginsenoside production in P. ginseng root cultures 24 . Also, Pseudomonas and Streptomyces spp. stimulated withaferin-A, 12-deoxywithstramonolide, and withanolide A production in in vitro W. somnifera plants 25 .

Conclusion
This research was conducted in an attempt to find a new producer of industrially valuable Amaryllidaceae alkaloids and a way to increase their biosynthesis in in vitro cultures of Leucojum aestivum. To the best of our knowledge, this is the first report on the possibility of galanthamine biosynthesis by an endophytic microorganism. Paenobacillus lautus strain PLV, isolated for the first time from L. aestivum in vitro plants showed great potential for the synthesis also others Amaryllidaceae alkaloids. Moreover, P. lautus strain PLV biosynthesized nine growth regulators and stimulated in vitro plant growth. Furthermore, it may elicit the biosynthesis of alkaloids that are not observed in untreated plants. The obtained results provide a new perspective on Amaryllidaceae alkaloids production, especially galanthamine. It will also be helpful in research on understanding the still unexplored mechanisms of galanthamine biosynthesis in 'plant' and 'bacterial' systems and the 'plant-bacterial' relationship.
Future research should focus on larger-scale biosynthesis of Amaryllidaceae alkaloids by P. lautus strain PLV using a fermenter, as well as on the optimization of elicitation in L. aestivum cultures.

Materials and methods
Isolation of endophytic bacteria from L. aestivum plants. L 10 . Afterwards, plant leaves and bulbs with visible bacteria were dissected into small pieces and transferred to a solid lysogeny broth medium (LB) 28 and incubated at 37 °C for 72 h.
To obtain single colonies, the bacteria were transferred to a fresh LB medium via streaking (using a platinum loop) and incubated at 37 °C for 24 h. The authors confirm that all methods used were performed in accordance with the relevant guidelines and legislation.

Molecular identification of endophytic bacteria.
Bacteria obtained from LB plates were stained with standard Gram technique for microscopic observation and to determine the DNA extraction method. A single bacterial colony was used to inoculate 10 mL of LB medium. After a 24 h incubation (37 °C on a shaker at 100 rpm) the cells were harvested by centrifugation at 6000 xg, for 10 min (Biofuge Stratos, Heraeus, Germany). Pellet was used for genomic DNA extraction using Genomic Maxi AX kit (A&A Biotechnology, Poland) according to manufacturer protocol with pre-treatment for Gram-positive bacteria including additional digestion steps with lysozyme and proteinase K. DNA concentration was assessed with NanoDrop2000c (Thermo Fisher Scientific, USA). The obtained genomic DNA of the endophytic bacteria was used for WGS (whole genome sequencing). The sequencing library was prepared commercially using Illumina's Nextera XT DNA library kit, and qual Raw sequencing reads were quality controlled using FastQC software (Babraham Bioinformatics), and filtered using Trim Galore software (Babraham Bioinformatics) along with adapter sequences removal. Cleaned reads were assembled into cotigs with Shovil v1.1.0 which is an ultra-fast implantation of Spades v3.14.0 algorithm 47 . The obtained contigs were finally polished using Pilon 1.23 software 48 and scaffolded using MeDuSa web server 49 against Paenibacillus lautus strain E7593-69 (GCA_003590055.1; ASM359005v1) genome. Scaffold belonging to plasmid was identified by Nucleotide BLAST against the reference genome sequence. Contigs statistics were retrieved using Quast v5.0.2 software 50 . The sequences were annotated during genome submission by the NCBI Prokaryotic Genome Annotation Pipeline (PGAP) 51 using Best-placed reference protein set (GeneMarkS-2+) v5.1. Taxonomic analysis of the isolated strain and search for the most similar spices were done using TYGS web server 52 . Both 16S rDNA (SSU) and whole-genome based phylogenic trees were constructed.

Preparation of bacterial material for alkaloid and growth regulator analysis. A single colony
of pure bacteria cultures was picked up with a pipette tip and inoculated with 50 mL of liquid LB medium in Erlenmeyer flasks (in 50 replicates). The liquid cultures were incubated for 24 h at 37 °C on a shaker at 100 rpm (Innova ® 42/42R New Brunswick™ incubator, Eppendorf, Germany). After this time, the cell suspension was centrifuged and the resulting bacterial cell pellet was lyophilized (FreeZone 6 Liter freeze dryer, Labconco, USA).
Elicitor preparation. One isolated colony of endophytic bacteria was transferred to 10 mL of LB liquid medium in a glass tube via pipette tip and incubated at 37 °C on a shaker at 100 rpm for 24 h. Bacterial quantification was performed using a spectrophotometer (NanoDrop 2000, Thermo Fisher Scientific, USA) at 600 nm. The bacterial culture was established to an optical density (OD) of 1.0 53,54 . Afterward, the bacteria were autoclaved and then centrifuged (6000 × g, 20 min). The supernatant was removed, and the cell sediment was resuspended in 10 mL of sterile distilled water and used for elicitation according to Lim et al. 55 . Elicitor treatments. L. aestivum plants 12 months old were obtained from somatic embryos and transferred to a liquid MS medium containing 5 µM of zeatin 39 . The experiment was carried out in the RITA ® bio-  9 . The identification of the alkaloids was performed by comparing the measured data with those of authentic compounds (galanthamine, lycorine) or with literature data as specified in the text. The alkaloids were quantified using  www.nature.com/scientificreports/ LC-MS equipment constituted by U3000-Dionex apparatus and micrOTOF Q ™ apparatus (Bruker Daltonics, Bruker, Bremen, Germany). An internal standard calibration method along with a nine-point calibration curve (R 2 = 0.99) using authentic galanthamine and lycorine were used for quantitative analysis of alkaloids. The analysis for quantification of alkaloids were repeated three times.  Data analysis. The results are expressed as mean values and standard deviation (SD). Statistical analysis of the experiment data was done with analysis of variance (ANOVA). Differences between the means were performed using Duncan's multiple range test at P < 0.05.

Data availability
The datasets generated during the current study are available from the corresponding author on reasonable request. The genome-wide shotgun design of the endophytic bacterium has been deposited with DDBJ/ENA/ GenBank as part of accession JAIFIS00000000. The version described in this paper is version JAIFIS010000000. The raw sequence data are available [https:// www. ncbi. nlm. nih. gov/ search/ all/? term= JAIFI S0100 00000].